Fshr is a critical component of the endocrine axis that controls gonad function, gametogenesis, and fertility. Characterization of the mechanisms regulating Fshr transcription provides the opportunity to reveal proteins involved in selective gene expression in Sertoli and granulosa cells and expand our understanding of factors regulating reproductive health. To date, transcriptional studies of Fshr have focused only on promoter and 5'flanking sequences. While these analyses are critical to our understanding of Fshr, it appears that regulatory sequences outside this region are important for Fshr transcriptional regulation. Fortunately, our ability to identify such regulatory sequences has advanced significantly due to the extensive progress in genome research, which provides a wealth of new information and resources. The proposed studies combine computation analysis, transcriptional assays, and transgenic approaches to further our understanding of the processes controlling Fshr transcription. In Aim I, studies are proposed to use mouse knockout models and chromatin immunoprecipitation to evaluate the in vivo roles of transcription factors implicated in Fshr regulation and expand our understanding of the mechanisms regulating transcription through the proximal promoter. In Aim II, comparative genomics, DNase I hypersensitivity mapping and transient transfection analysis will be used to identify distal regulatory regions within the Fshr locus. DNA/protein binding studies will be used to identify proteins associated with important regulatory elements. Aim III proposes to use transgenic mice for in vivo characterization of Fshr regulatory elements. An Fshr-containing yeast artificial chromosome (YAC) will be used to generate transgenic mice that will be characterized for expression in various tissues. Mutations within proposed regulatory elements will be generated in the YAC by homologous recombination and tested for correct expression in transgenic mice, to evaluate their role in vivo. The proposed studies will reveal regulatory elements and proteins needed for Fshr expression, leading not only to a better understanding of its transcriptional control but to the basic principles governing gonadal function, fertility, and generalized gene transcription. By advancing our understanding in these areas, we enhance our ability to evaluate matters of human reproductive health and to develop strategies to assist fertility problems and management.